| Autor: |
MacKenzie, C. Roger, Sharma, Vidhya, Brummell, David, Bilous, Doris, Dubuc, Ginette, Sadowska, Joanna, Young, N. Martin, Bundle, David R., Narang, Saran A. |
| Zdroj: |
Bio/Technology; April 1994, Vol. 12 Issue: 4 p390-395, 6p |
| Abstrakt: |
We have used a strategy of hybrid gene synthesis and constant domain shuffling to construct and functionally express in Escherichia coli genes encoding two anti–carbohydrate Fabs, one specific for a Brucella cell–surface polysaccharide and the second for the human blood group A determinant. Very similar VLamino acid sequences made possible the simultaneous synthesis of the two corresponding genes. A class switching approach was used in Fd and light chain gene assembly. The two independently synthesized VHgenes were fused to a previously made sequence encoding the Cγ11 domain as an alternative to synthesis of the natural Cγ2b1 and Cμ1 sequences. The VLgenes were initially coupled to a synthetic Cκgene. When these light chain and the above Fd genes, each preceded by the ompA signal sequence, were expressed from two–cistron DNA, yields of functional periplasmic Fab were low and, in each instance, limited by light chain availability. Replacement of the Cκdomains with a Cλ1domain resulted in a significant increase in the amount of soluble periplasmic light chain and functional Fab for both the Brucella and blood group A antibodies. The Cκand Cλ1forms of each of the Brucella and blood group A Fabs, with His5fusions at the C–termini of the Fd chains, were purified by immobilized metal affinity chromatography. For the blood group A antibody, it was shown by ELISA that precise engineering of the elbow region was essential for full activity of the hybrid light chain constructs, since a two residue increase in elbow length abolished antigen binding activity. The Brucella antibody tolerated the longer elbow sequence. Sequences in the Cλ1domain may result in increased yields of functional light chain by improving translocation across the cytoplasmic membrane or by reducing formation of periplasmic inclusion bodies. |
| Databáze: |
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